All living cells establish transmembrane electrochemical gradients with the help of primary ion pumps. Primary Na+ pumps have been discovered in many microorganisms of quite diverse phylogenic groups and a transmembrane circulation of Na+ ions may play a significant role in the physiology of several bacteria. The recent completion of many bacterial genome sequences revealed the presence of genes encoding a variety of sodium-dependent systems in many organisms, including some that were not known to have a primary sodium cycle of energy. This indicates that these bacteria can utilize Na+ as a coupling ion instead of, or in addition to, the H+ cycle. Although little is known about the role of the sodium cycle of energy in the physiology of Vibrio cholerae, the presence of a multitude of sodium-dependent systems encoded in the genome of this organism merits the investigation of its various Na+ pumps. In the present application we will construct and analyze defined mutants in Na+-extruding enzymes in V. cholerae as a model organism to gain further insights into this very complex and important part of bacterial physiology. We propose a comprehensive and systematic analysis of the components that contribute to bacterial sodium bioenergetics by using extensive genetic manipulations in combination with sophisticated bioenergetic measurements. Results of the proposed study will not only enhance our understanding of the general bioenergetic pathways in bacteria, but will form a solid basis for future investigations, by ourselves and others, of the molecular devices maintaining ion homeostasis in microorganisms. Sodium bioenergetics probably plays a role in both the environmental and pathogenic phases of the V. cholerae life cycle. Thus, our research has the potential for generating fundamental perspectives related to bacterial physiology and will be of value to understanding basic biological concepts related to V. cholerae ecology in the environment and in the lumen of hosts that will be applicable to a variety of bacterial species. [unreadable] [unreadable] [unreadable]